Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Abstract

Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X 2 and/or P2X 3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.Lys49 myotoxin | ATP release | pannexin | pain | purinergic receptor V enoms from spiders, snakes, cone snails, and scorpions have evolved to produce a vast pharmacopoeia of toxins that modulate receptor or channel function as a means of producing shock, hemolysis, paralysis, and pain (1, 2). As such, venoms from these animals represent a rich potential source of potent and selective toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. For example, toxins can directly target ion channels expressed by nociceptive sensory neurons to elicit or inhibit pain. Well-validated examples include toxins that target transient receptor potential (TRP) ion channels, acid-sensing ion channels (ASICs), and Na v channels (3-8). However, the "toxinome" is incredibly diverse, and there are many painproducing venoms for which the responsible toxins have not yet been identified.In addition to targeting receptors and channels directly, algogenic toxins could also promote the synthesis and/or release of metabolites, transmitters, or second messengers that activate primary afferent nociceptors. Although this could occur through indiscriminate actions of venom lipases and proteases that release cellular contents through tissue damage and cell death, more specific mechanisms may also exist. Their identification could reveal important new pathways by which exogenous or endogenous pain-producing agents mediate their effects.To identify novel pain-producing toxins, we have exploited cultured sensory neurons as a facile in vitro platform for unbiased screening of crude venoms, optimizing conditions to reduce nonspecific cell damage so as to reveal toxins having specific cellular effects. With this approach, we have identified a toxin (BomoTx) from the Brazilian lancehead pit viper (Bothrops moojeni) f...

Multifunctional Toxins in Snake Venoms and Therapeutic Implications: From Pain to Hemorrhage and Necrosis

“…This suggests that PLA2s contribute to an increase in arachidonic acid release from cell membranes and its availability to be processed by cyclooxygenase resulting in prostaglandin production (Verri et al, 2006). Corroborating this hypothesis, studies performed in rodents have demonstrated that PLA2s isolated from different snake venoms induced hyperalgesia mediated by biogenic amines, cytokines, prostaglandins, sympathomimetic amines, ATP, K + release, purinergic receptor activation, and glial cell activation (Nunez et al, 2001;Chacur et al, 2003Chacur et al, , 2004Zhang et al, 2017). Interestingly, the presence of a strong catalytic activity in the PLA2s is not essential for its nociceptive activity as observed by the nociceptive effects of PLA2s "Lys49" variants (Lomonte et al, 1994;Rong et al, 2016;Zhang et al, 2017).…”

“…A number of PLA2s exert strong myotoxic effects which often lead to severe necrosis (Harris and Maltin, 1982;Gutierrez and Ownby, 2003), and many of these toxins also promote inflammation, including edema formation, cytokine production and leukocyte recruitment, pain by inducing thermal allodynia and mechanical hyperalgesia, paralysis through block of neuromuscular transmission and intensify hemorrhage by inhibiting coagulation (Table 1) (Camara et al, 2003;Chacur et al, 2003;Camargo et al, 2008;Teixeira et al, 2011;Lomonte and Rangel, 2012;Harris and Scott-Davey, 2013;Casais-E-Silva et al, 2016;Costa et al, 2017;Zambelli et al, 2017b;Zhang et al, 2017). Neurotoxic effects caused by these toxins, as well as some of their proinflammatory effects, occurs via the modulation of pre-synaptic terminals as well as sensory nerveendings (Camara et al, 2003;Harris and Scott-Davey, 2013;Sribar et al, 2014;Zhang et al, 2017). The PLA2s pre-synaptic effects are characteristic of β-neurotoxins and target the motor nerve terminals at the neuromuscular junction (Sribar et al, 2014;Gutierrez et al, 2017).…”

“…The inflammation induced by PLA2s has non-neurogenic and neurogenic (substance-P dependent) components (Camara et al, 2003;Camargo et al, 2008;Costa et al, 2017;Zhang et al, 2017). The non-neurogenic component is mostly caused by the hydrolysis of membrane lipids that generate potent pro-inflammatory lipid mediators .…”

“…Furthermore, SVSPs and SVMPs induce hemostatic and cardiovascular effects as coagulopathy, hypotension and hemorrhage (Slagboom et al, 2017). Interestingly, some PLA2s, SVSPs, and SVMPs are also capable of triggering severe pain by modulating pain pathways through activation of ion channels, such as transient receptor potential vanilloid type 1 (TRPV1) and acid-sensing ion channel (ASIC) (Bohlen et al, 2011;Zhang et al, 2017); and/or by pain sensitization through inflammatory mediators (Zychar et al, 2010;Menaldo et al, 2013;Ferraz et al, 2015;Mamede et al, 2016;Zambelli et al, 2017b). The inflammation induced by the elapid and viper venoms is widely reported to produce pain or hyperalgesia in human and in experimental models (Hifumi et al, 2015;Bucaretchi et al, 2016;Kleggetveit et al, 2016;Mamede et al, 2016).…”

Structural venomics: evolution of a complex chemical arsenal by massive duplication and neofunctionalization of a single ancestral fold

“…Sequence diversity across taxa is minimal and no new activities have been reported for venom hyaluronidases 37 . In contrast, venom PLA 2 s are more diverse and they have acquired a range of new functions, some of which are independent of their catalytic activity 39,40 . There are only a few reports of lipases in animal venoms, but the H. infensa venom lipase has significant homology to those found to be active in wasp venoms 41,42 .…”